Regulation of gene expression is controlled at the level of RNA transcription. We propose in this application to investigate the regulation of expression of a set of inducible genes of Drosophila melanogaster termed the "heat shock genes". When Drosophila are shifted from the normal growth temperature of 25 degrees C to 37 degrees C, the synthesis of at least 7 proteins is induced. Since the response to heat shock begins quickly after the temperature shift, the system can be treated as a "classical" inducible system. Furthermore, most genes actively transcribed prior to heat shock are subsequently repressed, while a limited number continue to be transcribed after heat treatment. Therefore, the heat shock system offers a unique opportunity to examine induced, repressed and constitutive genes in parallel. Recombinant DNA technology is being exploited to isolate the induced heat shock genes as well as representatives of repressed and constitutive genes. Two approaches will be used in parallel to define regulatory regions responsible for control of transcription: (1) the primary DNA sequence of cloned genes will be determined with particular emphasis on putative regulatory regions near the point of initiation of transcription. We will determine if a comparison of the primary DNA sequence reveals similar features which may be required for the expression of all genes, and if other similarities exist among genes which respond to a certain stimulus in a particular manner. (2) We will begin an analysis of genetic mutants which contain alterations in putative regulatory regions constructed in vitro using recombinant DNA technology. The effect of the alterations on RNA transcription will be tested by adding purified DNA to either living cells or an in vitro reconstituted system and monitoring RNA transcription.